Optimal micropatterning of a collagen scaffold enhances the quality of nerve regeneration

Introduction Although the presence of a conduit between the stumps of a transected peripheral nerve is sufficient to induce regeneration, the microstructural, mechanical and compositional features of the tubular construct itself, and of any material inserted within the tube lumen, are very likely to significantly affect the quality of nerve regeneration. Pioneering studies on peripheral nerve regeneration made extensive use of biodurable conduits, such as silicone tubes and, recently, a wide variety of biodegradable materials, including synthetic and natural polymers, and different conduit geometries have been examined. Collagen has been identified as one of the most promising materials for the production of conduits, due to its biocompatibility and the observed enhanced cell attachment. Recently a new type of collagen neural guide, characterized by the presence of a radially micropatterned porosity, has been developed and patented [1]. In this study these novel tubes were tested in vivo in a rat model (10 mm gap sciatic nerve). The histological and electrophysiological results were encouraging for the design of a human study (sural nerve) to be performed in the near future, according to a protocol already approved by the ethical committee of the San Raffaele Hospital. Materials and Methods The collagen tubes were made starting from Type I collagen isolated from calf skin (SYMATESE Biomateriaux), which is EDQM certified. The tubular scaffolds were prepared using a five step process which includes: 1) preparation of a collagen-based slurry; 2) spinning of the slurry in a proper apparatus and rapid freezing of the centrifuged suspension in liquid nitrogen; 3) freeze drying of the solidified suspension to obtain the final tubular scaffold; 4) crosslinking (DHT) of the structure to confer suitable mechanical properties to the construct, and finally 5) sterilization (EtO) of the device. The resulting tubular scaffolds display a gradient in porosity and pore size along the tube radius, and a radially-oriented pore distribution [1]. In particular, the outer surface of the tube wall is protein-permeable but cell-impermeable, whereas the inner surface of the tube wall is cell-permeable. In this way, cells (e.g. myofibroblasts) can infiltrate the tube wall only from the inside. Results and Discussion The preclinical study performed on Sprague-Dawley rats demonstrated excellent results in terms of nerve regeneration and formation of stromal tissue. Indeed the novel micropatterned collagen tubes performed better than other randomly oriented collagen tubes already on the market. Notably, at 60 days post-operation in the regenerated mid-graft, the regenerated nerve had a normal histological composition regarding fiber size, number and myelination (Fig. 1). Four months after the surgery, the cMAP (compound Motor Action Potential) was recorded. For the rats treated with the novel conduit, the presence of the cMAP at the plantar muscles, following sciatic nerve stimulation above the site of nerve cutting, indicated a relatively early phase of nerve fibre regeneration and myelin repair after lesion. Fig. 1. Novel tubes at the mid graft 60 days after the implant. The collagen conduit was completely replaced by a normal nerve fascicle with normal perineurium and endoneurial tissue (a and b). Acknowledgements The authors acknowledge the Italian Ministry of University and Research (FIRB project TissueNet) for partial funding.